The invention relates to a novel tumor necrosis factor (TNF) homolog designated herein as TNF-related death ligand (TRDL).
Tumor necrosis factor (TNF), named for its ability to shrink tumors, is made by cells of the immune system and is a member of an emerging family of cytokines with important roles in immune regulation, inflammation and cancer. The family includes seven members, in addition to TNF, which share limited sequence homology that is confined to the C-terminal portion of the molecules. With exception of TNF-xcex2, each of these of ligands are type II membrane associated proteins that require cell surface presentation to elicit effects from corresponding target cells (Wiley, S. R., Schooley, K., Smolak, P. J., Din, W. S., Huang, C-P., Nicholl, J. K., Sutherland, G. R., Davis Smith, T., Rauch, C., Smith, C. A., and Goodwin, R. G. (1995) Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity. 3, 673-682). This cytokine family interacts with a growing list of target transmembrane receptors with complicated signaling strategies and often contradictory biological effects (Wiley, S. R., Schooley, K., Smolak, P. J., Din, W. S., Huang, C-P., Nicholl, J. K., Sutherland, G. R., Davis Smith, T., Rauch, C., Smith, C. A., and Goodwin, R. G. (1995) Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity. 3, 673-682).
The interaction of TNF-xcex1 with the TNFR-1 receptor typifies the biological diversity of the TNF ligand receptor family. Ligation of TNFR-1 can activate NF-xcexaB and elicit an inflammatory response in a variety of cell types (Beg, A. A. and Baltimore, D. (1996) An essential role for NF-xcexaB in preventing TNF-xcex1-induced cell death. Science. 274, 782-784. Wang, C-Y., Mayo, M. W., and Baldwin, A. S. Jr. (1996) TNF-and cancer therapy-induced apoptosis: potentiation by inhibition of NF-xcexaB. Science. 274, 784-787. VanAntwerp, D. J., Martin, S. J., Kafri, T., Green, D. R., and Verma, I. M. (1996) Suppression of TNF-xcex1-induced apoptosis by NF-xcexaB. Science. 274, 787-789).
Alternatively, TNFR-1 activation can also induce apoptosis (Pan, G., O""Rourke, K, Chinnaiyan, A. M., Gentz, R., Ebner, R., Ni, J., and Dixit, V. (1997) The receptor for the cytotoxic ligand TRAIL. Science. 276, 111-113). The mechanisms that regulate these two pathways and the final cellular outcome remain unclear, but offer interesting prospects for therapeutic intervention. The importance of this life death balance in human disease can be seen in the observation that TNF fails to efficiently kill many types of cancer cells (Wang, C-Y., Mayo, M. W., and Baldwin, A. S. Jr. (1996) TNF-and cancer therapy-induced apoptosis: potentiation by inhibition of NF-xcexaB. Science. 274, 784-787. Baichwal, V. R., and Baeuerle, P. A. (1997) Apoptosis: Activate NF-xcexaB or die? Current Biology. 7, R94-R96). Recent evidence suggests that TNF undermines its own killing powers by activating NF-xcexaB, a key molecule that can block the apoptosis pathway (Beg, A. A. and Baltimore, D. (1996) An essential role for NF-xcexaB in preventing TNF-xcex1-induced cell death. Science. 274, 782-784. Wang, C-Y., Mayo, M. W., and Baldwin, A. S. Jr. (1996) TNF-and cancer therapy-induced apoptosis: potentiation by inhibition of NF-xcexaB. Science. 274, 784-787. VanAntwerp, D. J., Martin, S. J., Kafri, T., Green, D. R., and Verma, I. M. (1996) Suppression of TNF-xcex1-induced apoptosis by NF-xcexaB. Science. 274, 787-789. Liu, Z-G., Hsu, H., Goeddel, D., and Karin, M. (1996) Dissection of the TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-xcexaB activation prevents cell death. Cell. 87, 565-576. Wu, M., Lee, H., Bellas, R. E., Schauer, S. L., Arsura, M., Katz, D., FitzGerald, M. J., Rothstein, T. L., Sherr, D. H., and Sonenshein, G. E. (1996) Inhibition of NF-xcexaB/Rel induces apoptosis of murine B cells. EMBO J. 15, 4682-4690). This blockade may render tumor cells resistant to immune surveillance and confound chemotherapeutic approaches that rely on tumor cell apoptosis. Disruption of this protective mechanism may, therefore, sensitize cells to TNF mediated killing (Beg, A. A. and Baltimore, D. (1996) An essential role for NF-xcexaB in preventing TNF-xcex1-induced cell death. Science. 274, 782-784. Wang, C-Y., Mayo, M. W., and Baldwin, A. S. Jr. (1996) TNF-and cancer therapy-induced apoptosis: potentiation by inhibition of NF-xcexaB. Science. 274, 784-787. VanAntwerp, D. J., Martin, S. J., Kafri, T., Green, D. R., and Verma, I. M. (1996) Suppression of TNF-xcex1-induced apoptosis by NF-xcexaB. Science. 274, 787-789).
Recent advances in the understanding of TNF signaling have elucidated discrete molecular targets for potential blockade of NF-xcexaB activation and apoptosis in cells responding to TNF (Beg, A. A. and Baltimore, D. (1996) An essential role for NF-xcexaB in preventing TNF-xcex1-induced cell death. Science. 274, 782-784. Wang, C-Y., Mayo, M. W., and Baldwin, A. S. Jr. (1996) TNF-and cancer therapy-induced apoptosis: potentiation by inhibition of NF-xcexaB. Science. 274, 784-787. VanAntwerp, D. J., Martin, S. J., Kafri, T., Green, D. R., and Verma, I. M. (1996) Suppression of TNF-xcex1-induced apoptosis by NF-xcexaB. Science. 274, 787-789. Wu, M., Lee, H., Bellas, R. E., Schauer, S. L., Arsura, M., Katz, D., FitzGerald, M. J., Rothstein, T. L., Sherr, D. H., and Sonenshein, G. E. (1996) Inhibition of NF-xcexaB/Rel induces apoptosis of murine B cells. EMBO J. 15, 4682-4690). These responses are facilitated by the recruitment of signaling proteins to activated TNF receptors. Some of these signaling proteins, including TRADD (TNFR1-associated death domain protein), TRAF2 (TNFR-associated protein-2) and RIP (receptor interacting protein kinase) appear to initiate activation of NF-xcexaB. Chinnaiyan, A. M., O""Rourke, K, Yu, G-L., Lyons, R. H., Garg, M., Duan, D. R., Xing, L., Gentz, R., Ni, J., and Dixit, V. M. (1996) Signal transduction by DR3, a death domain-containing receptor related to TNFR-1 and CD95. Science. 274, 990-992. Recruitment of other proteins, including FADD (Fas-associated death domain protein) and FLICK (FADD-like interleukin converting enzyme) induces apoptosis (Boldin, M. P., Goncharov, T. M., Goltsev, Y. V., and Wallach, D. (1996) Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1-and TNF receptor-induced cell death. Cell. 85, 803-815. Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O""Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E., and Dixit, V. (1996) FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell. 85, 817-827).
This emerging biology has created considerable interest among researchers for potential therapeutic intervention, particularly in cancer, inflammatory diseases, and neurodegenerative disorders. Thus, it will be clear to the skilled artisan that there is a continuing need for novel members of the TNF family of inflammatory cytokines involved in apoptosis and NF-xcexaB activation.
Wiley et al., Immunity 3, 673-682 (1995).
Beg, A. A. and D. Baltimore, Science 274, 782-784 (1996).
Wang et al., Science 274, 784-787 (1996).
VanAntwerp et al., Science 274, 787-789 (1996).
Pan et al., Science 276, 111-113 (1997).
Baichwal et al., Current Biology 7, R94-R96 (1997).
Liu et al., Cell 87, 565-576 (1996).
Wu et al., EMBO J 15, 4682-4690 (1996).
Chinnaiyan et al., Science 274, 990-992 (1996).
Boldin et al., Cell 85, 803-815 (1996).
Muzio et al., Cell 85, 817-827 (1996).
Altschul et al., J. Mol. Bio. 215, 403-410 (1990).
Idziorek et al., J. Immuno. Meth. 185:249-258 (1995).
X. M. Wang, et al., Human Immunol 37: 264 (1993).
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a novel TNF homolog designated herein as TRDL, or a fragment thereof. TRDL polypeptides of the invention include two alternative splice variants of TRDL, designated herein as TRDL-11 and TRDL-14, which have the amino acid sequences shown in FIGS. 3 and 4 (SEQ ID NO:2 and SEQ ID NO:4, respectively). Preferred fragments of TRDL include the extracellular portion of TRDL-11 and of TRDL-14.
In a preferred embodiment, the nucleic acid molecules comprise a polynucleotide having the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3. In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent conditions to a polynucleotide encoding TRDL-11 or TRDL-14, or fragments thereof.
The present invention also provides vectors comprising the isolated nucleic acid molecules of the invention, host cells into which such vectors have been introduced, and recombinant methods of obtaining a TRDL polypeptide comprising culturing the above-described host cell and isolating the TRDL polypeptide.
In another aspect, the invention provides isolated TRDL polypeptides, as well as fragments thereof. In a preferred embodiment, the TRDL polypeptides have the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. Isolated antibodies, both polyclonal and monoclonal, that bind specifically to TRDL polypeptides are also provided.
The invention also provides a method for the identification of cells having a high affinity receptor for TRDL, the method comprising:
(a) labeling an isolated TRDL polypeptide
(b) contacting the labeled TRDL polypeptide obtained in step (a) with cells of a mammalian cell line;
(c) washing the cells obtained in step (b) to remove unbound TRDL; and
(d) determining the presence of the labeled TRDL polypeptide in the washed cells obtained in step (c);
whereby the presence of labeled TRDL polypeptide in the washed cells obtained in step (c) indicates the presence of a high affinity receptor for TRDL on the cells.
In another embodiment, the invention relates to a method for the identification of an agent which inhibits the binding of TRDL to its receptor, the method comprising:
(a) labeling an isolated TRDL polypeptide;
(b) contacting the labeled TRDL polypeptide obtained in step (a)
xe2x80x83with cells of a mammalian cell line
(i) in the presence of a test agent; and
(ii) in the absence of a test agent;
(c) washing the cells
(i) obtained in step (b)(i); and
(ii) obtained in step (b)(ii) to remove unbound TRDL;
(d) determining the amount of labeled TRDL polypeptide in
(i) the washed cells obtained in step (c)(i); and
(ii) the washed cells obtained in step (c)(ii); and
(e) comparing the amount of labeled TRDL polypeptide determined in step (d)(i) to that determined in (d)(ii);
whereby a lower amount of labeled TRDL polypeptide in sample (d)(i) than in sample (d)(ii) indicates that said agent inhibited the binding of TRDL to its receptor.
In another embodiment, the invention relates to a method for the identification of an agent which enhances the binding of TRDL to its receptor, the method comprising:
(a) labeling an isolated TRDL polypeptide;
(b) contacting the labeled TRDL polypeptide obtained in step (a)
xe2x80x83with cells of a mammalian cell line
(i) in the presence of a test agent; and
(ii) in the absence of a test agent;
(c) washing the cells
(i) obtained in step (b)(i); and
(ii) obtained in step (b)(ii) to remove unbound TRDL;
(d) determining the amount of labeled TRDL polypeptide in
(i) the washed cells obtained in step (c)(i); and
(ii) the washed cells obtained in step (c)(ii); and
(e) comparing the amount of labeled TRDL polypeptide determined in step (d)(i) to that determined in (d)(ii);
whereby a higher amount of labeled TRDL polypeptide in sample (d)(i) than in sample (d)(ii) indicates that said agent has enhanced the binding of TRDL to its receptor.
In another embodiment, the invention provides A method for the identification of a cell line that undergoes apoptosis upon interaction with TRDL, the method comprising:
(a) dividing the cells of a culture of a mammalian cell line into a test culture and a control culture;
(b) contacting a TRDL polypeptide with the test culture of step (a);
(c) determining the quantity of cells of
(i) the test culture obtained in step (b); and
(ii) the control culture of step (a);
xe2x80x83that have undergone apoptosis; and
(d) comparing the quantity of cells determined to have undergone apoptosis in the test culture of step (c)(i) with the quantity of cells determined to have undergone apoptosis in the control culture of step (c)(ii);
whereby a determination that the quantity of cells having undergone apoptosis in said test culture is higher than in said control culture indicates that said mammalian cell line undergoes apoptosis upon interaction with TRDL.
In yet another embodiment, the invention provides a method for the identification of an agent capable of inhibiting TRDL-mediated induction of apoptosis, said method comprising
(a) determining the quantity of cells that have undergone apoptosis in a test culture and a control culture, wherein said test culture comprises mammalian cells capable of undergoing apoptosis upon interaction with TRDL which have been contacted with a TRDL polypeptide in the presence of a test agent, and said control culture comprises mammalian cells capable of undergoing apoptosis upon interaction with TRDL which have been contacted with a TRDL polypeptide in the absence of a test agent; and
(b) comparing the quantity of cells determined to have undergone apoptosis in the test culture of step (a) with the quantity of cells determined to have undergone apoptosis in the control culture of step (a);
whereby a determination that the quantity of cells having undergone apoptosis in said test culture is lower than in said control culture indicates that said test agent inhibits TRDL-mediated induction of apoptosis.
In yet another embodiment, the invention provides a method for the identification of an agent capable of enhancing TRDL-mediated induction of apoptosis, said method comprising
(a) determining the quantity of cells that have undergone apoptosis in a test culture and a control culture, wherein said test culture comprises mammalian cells capable of undergoing apoptosis upon interaction with TRDL which have been contacted with a TRDL polypeptide in the presence of a test agent, and said control culture comprises mammalian cells capable of undergoing apoptosis upon interaction with TRDL which have been contacted with a TRDL polypeptide in the absence of a test agent; and
(b) comparing the quantity of cells determined to have undergone apoptosis in the test culture of step (a) with the quantity of cells determined to have undergone apoptosis in the control culture of step (a);
whereby a determination that the quantity of cells having undergone apoptosis in said test culture is higher than in said control culture indicates that said test agent enhances TRDL-mediated induction of apoptosis.
In yet another embodiment, the invention provides a method for the identification of an agent capable of inhibiting TRDL-mediated prevention of apoptosis, said method comprising:
(a) determining the quantity of cells that have undergone apoptosis in a test culture and a control culture, wherein said test culture comprises mammalian cells that are prevented from undergoing apoptosis upon interaction with TRDL which have been contacted with a TRDL polypeptide in the presence of a test agent, and said control culture comprises mammalian cells that are prevented from undergoing apoptosis upon interaction with TRDL which have been contacted with a TRDL polypeptide in the absence of a test agent; and
(b) comparing the quantity of cells determined to have undergone apoptosis in the test culture of step (a) with the quantity of cells determined to have undergone apoptosis in the control culture of step (a);
whereby a determination that the quantity of cells having undergone apoptosis in said test culture is higher than in said control culture indicates that said test agent inhibits TRDL-mediated prevention of apoptosis.
In yet another embodiment, the invention provides a method for the identification of an agent capable of enhancing TRDL-mediated prevention of apoptosis, said method comprising
(a) determining the quantity of cells that have undergone apoptosis in a test culture and a control culture, wherein said test culture comprises mammalian cells that are prevented from undergoing apoptosis upon interaction with TRDL which have been contacted with a TRDL polypeptide in the presence of a test agent, and said control culture comprises mammalian cells that are prevented from undergoing apoptosis upon interaction with TRDL which have been contacted with a TRDL polypeptide in the absence of a test agent; and
(b) comparing the quantity of cells determined to have undergone apoptosis in the test culture of step (a) with the quantity of cells determined to have undergone apoptosis in the control culture of step (a);
whereby a determination that the quantity of cells having undergone apoptosis in said test culture is lower than in said control culture indicates that said test agent enhances TRDL-mediated prevention of apoptosis.